The epidermis is the first line of defense against microbial infections of the skin. We recently reported that human corneal epithelial cells as well as human and murine keratinocytes are capable of expressing a family of recently described receptors termed Toll-like receptors (TLRs). To date, most published studies have examined the expression and function of TLR in leukocytes. Much less is known about the role of TLR in non-leukocytes such as keratinocytes. In this application we will utilize both an in vitro and in vivo murine model system to test the following hypotheses : 1) Keratinocyte TLR2 and TLR4 play an essential role in host cutaneous defenses to the gram (+) and gram (-) pathogens Staphylococcus aureus and Pseudomonas aeruginosa. 2) These protective anti-bacterial responses are mediated by activation of keratinocyte TLR2 and TLR4 by specific components of Staphylococcus aureus and Pseudomonas aeruginosa that initiate the local expression and production of potent antimicrobial innate immune effector proteins. 3) Defects in the expression or function of keratinocyte TLR2 and TLR4 will result in altered epidermal innate immune protective responses to S. aureus and P. aeruginosa that may lead to a progressive cutaneous infectious process causing increased morbidity and even death as a result of systemic spread of these bacterial pathogens. These hypotheses will be tested in the following specific aims: Specific Aim #1: To examine the expression and function of TLR2 and TLR4 in cultured murine keratinocytes in response to gram (+) and gram (-) bacteria, structural components derived from these bacteria, as well as selected proinflammatory and immunosuppressive agents. Specific Aim #2: To assess selected TLR2 and TLR4 mediated innate immune responses in cultured murine keratinocytes in response to pathogenic gram (+) and gram (-) bacteria and structural components derived from these bacteria. Specific Aim #3: To determine the in vivo role of murine keratinocyte TLR2 and TLR4 in mediating host protective epidermal innate immune responses to gram (+) and gram (-) bacterial infection and bacterial-derived components. Murine keratinocyte responses to pathogenic bacteria will be compared to responses to cutaneous commensal bacteria and confirmed in human keratinocytes. We believe that our studies will lead to novel insights into cutaneous innate immunity and the management of a wide range of inflammatory and infectious diseases in the skin.